The overall goal of this 5 year research plan is to characterize the immunomodulatory activity of newly isolated endogenous cannabinoid receptors ligands. These studies are based on the recent discovery of cannabinoid receptors, CB1 and CB2, and putative endogenous cannabinoid receptor ligands, anandamide (Ana) and 2-arachidonyl glycerol (2-Ara-Gl). Although cannabinoid receptors (CB1) were initially identified in the brain, careful examination of other tissues has revealed the expression of CB1 and more recently a second type of cannabinoid receptor, CB2, in spleen. Our laboratory was the first to identify the existence of functional cannabinoid receptors associated with the immune system. Although the biological role for these receptors is unclear, a significant amount has been learned about their structure and the signal transduction events which follow their interaction with ligands. Both CB1 and CB2 are 6-protein coupled receptors which effectively inhibit adenylate cyclase in B- and T-lymphocytes. We have consistently observed a strong correlation between the ability of cannabinoids to inhibit adenylate cyclase and altered immune function. Preliminary findings from our laboratory show that 2-Ara-Gl, originally isolated from canine gut, inhibits adenylate cyclase in mouse spleen cells as well as proliferative responses to defined mitogenic stimuli in a manner similar to delta9-THC. In contrast, Ana, which thus far has been identified solely in brain tissue, has very modest effects on immune function and adenylate cyclase activity on lymphoid cells. These findings and with those presented in this proposal, suggest that 2-Ara-Gl may be a novel but relevant immunoregulatory factor which functions by a similar if not identical mechanism to plant-derived and synthetic cannabinoids. Based on these finds, a multifacted approach will be used to test the following HYPOTHESIS: The immune 7 system is regulated in part through the release of endogenous cannabinoid receptor ligands. These ligands are immunosuppressive and mediate their effects through G-protein coupled receptors which are negatively coupled to adenylate cyclase therefore producing a decrease in intracellular cAMP in lymphoid cells. This hypothesis will be tested using the following specific aims: (1) To comprehensively characterize the immunoregulatory activity of 2-Ara-Gl and Ana using defined immune function assays; (2) To establish the binding characteristics of Ana and 2-Ara-Gl in mouse spleen cells and in purified spleen cell subpopulations; (3) To characterize the modulation of adenylate cyclase by 2-Ara-Gl in mouse spleen cells; and (4) To establish whether the immunological and biochemical changes induced by 2-Ara-Gl are mediated through CB1, CB2 or both. We believe that the successful completion of these specific aims will provide important insight into the role of cannabinoid receptors on immune function and the regulatory actions mediated by their ligands.